U.S. patent number 6,548,080 [Application Number 10/124,424] was granted by the patent office on 2003-04-15 for method for partially demineralized cortical bone constructs.
This patent grant is currently assigned to Musculoskeletal Transplant Foundation. Invention is credited to Arthur A. Gertzman, Moon Hae Sunwoo.
United States Patent |
6,548,080 |
Gertzman , et al. |
April 15, 2003 |
Method for partially demineralized cortical bone constructs
Abstract
The invention is directed toward a sterile bone structure for
application to a bone defect site to promote new bone growth at the
site comprising a partially demineralized cortical bone structure,
said bone structure comprising a cross sectional surface are
ranging from 85% to 95% of the original bone surface area before
demineralization with the remaining partially demineralized
cortical bone structure having an outer demineralized layer ranging
in thickness from about 0.05 mm to about 0.14 mm and a mineralized
core.
Inventors: |
Gertzman; Arthur A. (Stony
Point, NY), Sunwoo; Moon Hae (Old Tappan, NJ) |
Assignee: |
Musculoskeletal Transplant
Foundation (Edison, NJ)
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Family
ID: |
27101914 |
Appl.
No.: |
10/124,424 |
Filed: |
April 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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739214 |
Dec 19, 2000 |
6432436 |
|
|
|
677891 |
Oct 3, 2000 |
6458375 |
|
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Current U.S.
Class: |
424/423; 424/400;
424/422; 424/549; 424/94.1; 514/785; 514/801; 514/802 |
Current CPC
Class: |
A61F
2/28 (20130101); A61L 27/3608 (20130101); A61L
27/365 (20130101); A61L 27/3683 (20130101); A61L
27/3691 (20130101); A61L 27/3847 (20130101); A61L
31/005 (20130101); A61B 17/86 (20130101); A61F
2/3094 (20130101); A61F 2/446 (20130101); A61F
2/447 (20130101); A61F 2002/2817 (20130101); A61F
2002/2839 (20130101); A61F 2002/30059 (20130101); A61F
2002/302 (20130101); A61F 2002/3023 (20130101); A61F
2002/30843 (20130101); A61F 2002/30892 (20130101); A61F
2002/30894 (20130101); A61F 2230/0065 (20130101); A61F
2230/0069 (20130101); A61F 2310/00293 (20130101); A61L
2430/02 (20130101); Y10S 514/801 (20130101); Y10S
514/802 (20130101) |
Current International
Class: |
A61F
2/28 (20060101); A61L 27/00 (20060101); A61L
27/36 (20060101); A61L 31/00 (20060101); A61B
17/86 (20060101); A61B 17/68 (20060101); A61F
2/30 (20060101); A61F 2/00 (20060101); A61F
2/44 (20060101); A61F 002/28 (); A61F 002/30 ();
A61F 002/32 (); A61F 002/38 (); A61F 002/40 () |
Field of
Search: |
;424/422,94.1,423,549
;514/785,801,802 ;623/16 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Hallfeldt et al., Sterilization of Partially Demineralized Bone
Matrix: The Effects of Different Sterilization Techniques on
Osteogenetic Properties; Journal of Surgical Research 59, 1995, pp.
614-620, vol. 59.* .
Hallfeldt et al., Sterilization of Partially Demineralized Bone
Matrix: The Effects of Different Sterilization Techniques on
Osteogenetic Properties; Journal of Surgical Research 59, 1995, pp.
614-620, vol. 59..
|
Primary Examiner: Page; Thurman K.
Attorney, Agent or Firm: Hale; John S. Gipple & Hale
Parent Case Text
RELATED APPLICATION
The present invention is divisional application of U.S. patent
application Ser. No. 09/739,241 now U.S. Pat. No. 6,432,436, filed
Dec. 19, 2000 which is a continuation-in-part of U.S. patent
application Ser. No. 09/677,891 now U.S. Pat. No. 6,458,375, filed
Oct. 3, 2000.
Claims
What we claim is:
1. A method for partially demineralizing a previously shaped
cortical bone structure comprising the steps of: a) soaking said
previously shaped cortical bone structure in an acid solution for a
time period at a temperature less than about 30.degree. C. to
remove a layer of the cortical bone structure and produce a
demineralized layer on the cortical bone structure ranging from
about 0.05 mm to about 0.08 mm with the remaining area comprising
mineralized bone, the cortical bone structure after partial
demineralization being rigid and maintaining substantially its
original mechanical strength; b) agitating the acid solution and
immersed cortical bone structure; c) removing the cortical bone
structure from the acid solution and washing the cortical bone
structure until the wash discard is at about a neutral pH; d)
packaging the cortical bone structure in a moisture permeable
container; and e) lyophiizig the cortical bone structure.
2. A method as claimed in claim 1 wherein said acid solution is
hydrochloric acid ranging in acid concentrations from about 0.1N to
about 2.0N HCl.
3. A method as claimed in claim 1 wherein said acid solution
consists of a group consisting of hydrochloric acid, sulfuric acid,
phosphoric acid, mineral acids and organic acids.
4. A method as claimed in claim 1 wherein said acid solution is a
cheating agent ethylene diamine tetra acetic acid.
5. A method for partially dernineralizing a previously shaped hone
structure comprising the steps of: a) soaking said previously
shaped cortical bone structure having a thickness greater than 1.5
mm in an acid solution for 30 to 90 minutes at ambient temperature
to remove a layer of the cortical bone structure ranging from 0.12
mm to 0.40 mm of the surface area and produce bone structure with a
mineralized center section and a demineralized layer around said
mineralized center section ranging in thickness from about 0.08 mm
to about 0.14 mm bone, the cortical bone structure after
demineralization being rigid; b) simultaneously agitating the acid
solution and immersed cortical bone structure by stirring same; c)
removing the partially demineralized cortical bone structure from
the acid solution and washing the cortical bone structure with
sterile pure water until the wash discard is at about a neutral pH;
d) lyophilizing the cortical bone structure; and e) packaging the
cortical bone structure in a moisture permeable container.
6. A method as claimed in claim 5 wherein said acid solution is
hydrochloric acid ranging in acid concentrations from about 0.1N to
about 2.0N HCl.
7. A method as claimed in claim 5 wherein said acid solution
consists of a group consisting of hydrochloric acid, sulfuric acid,
phosphoric acid, mineral acids and organic acids.
8. A method as claimed in claim 5 wherein said acid solution is a
cheating agent ethylene diamine tetra acetic acid.
9. A method as claimed in claim 5 wherein said acid solution has a
temperature ranging from between 4.degree. C. and 30.degree. C.
10. A method for partially demineralizing a previously shaped bone
structure comprising the steps of: a) soaking a formed cortical
bone structure in an aqueous antibiotic solution; b) placing the
soaked cortical bone structure in an aqueous detergent at about 95
degrees F; c) applying ultrasonic energy to enhance penetration of
said detergent; d) washing the shaped cortical bone structure for
at least 60 minutes in an alcohol/water solution; e) soaking a
formed cortical bone structure in an acid solution for 15 to 30
minutes to remove a layer of the cortical bone structure and
produce a demineralized layer ranging from about 0.05 mm to 0.08 mm
in thickness bone, the cortical bone structure after
deminerazzation being rigid; f) agitating the acid solution with
said immersed cortical bone structure; g) removing the cortical
bone structure from the acid solution and washing the cortical bone
structure until the wash discard is at about a neutral pH; h)
lyophilizing the cortical bone structure; and i) packaging the
cortical bone structure in a moisture permeable container.
11. A method as claimed in claim 10 wherein said acid solution
consists of a group consisting of hydrochloric acid, sulfuric acid,
phosphoric acid, mineral acids and organic acids.
12. A method as claimed in claim 10 wherein said acid solution is a
cheating agent ethylene diarine tetra acetic acid.
13. A method as claimed in claim 10 wherein said acid solution is
hydrochloric acid having a concentration ranging from about 0.1N to
about 2.0N HCl and said acid soaking takes place at a temperature
ranging from between 4.degree. C. and 30.degree. C.
14. A method as claimed in claim 10 wherein said aqueous antibiotic
solution is Gentamysin.
15. A method as claimed in claim 10 wherein after step g), BMP is
added to the demineralized layer.
16. A method as claimed in claim 10 wherein after step g),
antimicrobial and/or antibiotics selected from the group consisting
of erythromycin, bacitracin, neomycin, penicilln, polymyxin B,
tetracycline, viomycin, chloromycetin and streptomycin, cefazolin,
ampicillin, azactam, tobramycin, clindamycin and gentamycin is
added to the demineralized layer.
17. A method for partially demineralizing a previously shaped bone
structure comprising the steps of: a) soaking a formed cortical
bone structure in an aqueous antibiotic solution; b) placing the
soaked cortical bone structure in an aqueous detergent at about 95
degrees F; c) applying ultrasonic energy to enhance penetration of
said detergent; d) washing the shaped cortical bone structure for
at least 60 minutes in an alcohol/water solution; e) soaking a
formed cortical bone structure in an acid solution for 15 to 30
minutes to remove a layer of the cortical bone structure and
produce a demineralized layer ranging from about 0.05 mm to 0.08 mm
in thickness bone, the cortical bone structure after
demineralization being rigid; f) agitating the acid solution with
an immersed cortical bone structure; g) removing the cortical bone
structure from the acid solution and washing the cortical bone
structure until the wash discard is at about a neutral pH; h)
adding a soluble silver compound to the demineralized layer; i)
lyophilizing the cortical bone structure; and j) packaging the
cortical bone structure in a moisture permeable container.
18. A method as claimed in claim 17 wherein said soluble silver
compound contains silver in a range of 10 to 10,000 parts per
million.
19. A method for partially dernineralizing a previously shaped
cortical bone structure comprising the steps of: a) soaking said
previously shaped cortical bone structure in an acid solution for a
time period at a temperature less than about 30.degree. C. to
remove a layer of the cortical bone structure while maintaining a
cross sectional area of the cortical bone structure ranging from
about 85% to about 95% and produce a demineralized layer on the
cortical bone structure ranging from about 0.05 mm to about 0.08 mm
with the remaining area comprising mineralized bone, the cortical
bone structure after partial demineralization being rigid and
maintaining suitable compression and bending strength; b) agitating
the acid solution and immersed cortical bone structure; c) removing
the cortical bone structure from the acid solution and washing the
cortical bone structure until the wash discard is at about a
neutral pH; d) packaging the cortical bone structure in a moisture
permeable container; and e) lyophilizing the cortical bone
structure.
Description
FIELD OF INVENTION
The present invention is generally directed toward a surgical bone
product and more specifically is a shaped partially demnineralized
allograft bone device or construct with a mineralized central
section.
BACKGROUND OF THE INVENTION
The use of substitute bone tissue dates back around 1800. Since
that time research efforts have been undertaken toward the use of
materials which are close to bone in composition to facilitate
integration of bone grafts. Development have taken place in the use
of grafts of a mineral nature such as corals, hydroxyapatites,
ceramics or synthetic materials such as biodegradable polymer
materials. Surgical implants should be designed to be biocompatible
in order to successfully perform their intended function.
Biocompatibility may be defined as the characteristic of an implant
acting in such a way as to allow its therapeutic function to be
manifested without secondary adverse affects such as toxicity,
foreign body reaction or cellular disruption.
Human allograft tissue is widely used in orthopaedic, neuro-,
maxilofacial, podiatric and dental surgery. The tissue is valuable
because it is strong, biointegrates in time with the recipient
patient's tissue and can be shaped either by the surgeon to fit the
specific surgical defect or shaped commercially in a manufacturing
environment. Contrasted to most synthetic absorbable or
nonabsorbable polymers or metals, allograft tissue is bioinert and
integrates with the surrounding tissues. Allograft bone occurs in
two basic forms; cancerous and cortical. Cortical bone is a highly
dense structure comprised of triple helix strands of collagen
fiber, reinforced with hydroxyapatite. The cortical bone is a
compound structure and is the load bearing component of long bones
in the human body. The hydroxyapatite component is responsible for
the high compressive strength of the bone while the collagen fiber
component contributes in part to torsional and tensile
strength.
Many devices of varying shapes and forms can be fabricated from
allograft cortical tissue by machining and surgical implants such
as pins, rods, screws, anchors, plates, intervertebral spacers and
the like have been made and used successfully in human surgery.
These engineered shapes are used by the surgeon in surgery to
restore defects in bone to the bone's original anatomical shape.
This treatment is well known in the art and is commercially
available as demineralized bone.
Allograft bone is a logical substitute for autologous bone. It is
readily available and precludes the surgical complications and
patient morbidity associated with obtaining autologous bone as
noted above. Allograft bone is essentially a collagen fiber
reinforced hydroxyapatite matrix containing active bone morphogenic
proteins (BMP) and can be provided in a sterile form The
demineralized form of allograft bone is naturally both
osteoinductive and osteoconductive. The demineralized allograft
bone tissue is fully incorporated in the patient's tissue by a well
established biological mechanism. It has been used for many years
in bone surgery to fill the osseous defects previously
discussed.
Demineralized allograft bone is usually available in a lyophilized
or freeze dried and sterile form to provide for extended shelf
life. The bone in this form is usually very coarse and dry and is
difficult to manipulate by the surgeon. One solution to use such
freeze dried bone has been provided in the form of a commercially
available product, GRAFTON.RTM., a registered trademark of
Osteotech Inc., which is a simple mixture of glycerol and
lyophilized, demineralized bone powder of a particle size in the
range of 0.1 cm to 1.2 cm as is disclosed in U.S. Pat. No.
5,073,373 issued Dec. 17, 1991 forming a gel. Similarly U.S. Pat.
No. 5,290,558 issued Mar. 1, 1994, discloses a flowable
demineralized bone powder composition using a osteogenic bone
powder with large particle size ranging from about 0.1 to about 1.2
cm mixed with a low molecular weight polyhydroxy carrier possessing
from 2 to about 18 carbons comprising a number of classes of
different compounds such as monosaccharides, disaccharides, water
dispersible oligosaccharides and polysaccharides.
A recent version of GRAFTON.RTM. product uses relatively large
demineralized particles in the carrier to create a heterogenous
mixture which provides body or substance to the composition. This
material is useful in filling larger defects where some degree of
displacement resistance is needed by the filler.
The advantages of using the bone particle sizes as disclosed in the
U.S. Pat. Nos. 5,073,373 and 5,290,558 patents previously discussed
were compromised by using bone lamellae in the shape of threads or
filaments having a median length to median thickness ratio of about
10:1 and higher while still retaining the low molecular weight
glycerol carrier. This later prior art is disclosed in U.S. Pat.
No. 5,314,476 issued May 24, 1994 and U.S. Pat. No. 5,507,813
issued Apr. 16, 1996 and the tissue forms described in these
patents are known commercially as the GRAFTON.RTM. Putty and Flex,
respective
The combination of natural cortical bone with very desirable
mechanical strength and the addition of synthetic (recombinant)
BMPs provides a superior form of tissue for surgical use retaining
all of the mechanical properties of the cortical component and the
accelerated healing offered by the BMP's.
U.S. Pat. No. 5,972,368 issued on Oct. 26, 1999 discloses the use
of cortical contructs (e.g. a cortical dowel for spinal fusion)
which are cleaned to remove all of the cellular material, fat, free
collagen and non-collagenous protein leaving structural or bound
collagen which is associated with bone mineral to form the
trabecular struts of bone. It is stated that the natural
crystalline structure of bone is maintained without the risk of
disease transmission or significant imnmunogenicity. Thus the
shaped bone is processed to remove associated non-collagenous bone
proteins while maintaining native bound collagen materials and
naturally associated bone minerals. Recombinant BMP-2 is then
dripped onto the dowel surface. It could also be added to the
cortical bone by soaking in the BMP-2 solution. As noted, this
reference teaches the removal of all non-collagenous bone proteins
which necessarily include all the naturally occurring BMP 's and
relies upon the addition of recombinant BMP-2 in a specific and
empirically determined concentration. The naturally occurring BMP's
are present in a concentration unique for each specific BMP protein
and has been optimized by nature. The '368 patent teaches complete
removal of the natural BMP's by demineralization and relies solely
on the added rhBNP's. The surface of a machined cortical bone
surface is characterized by a wide variety of openings resulting
from exposure by the machining process of the Haversian canals
present throughout cortical bone. These canals serve to transport
fluids throughout the bone to facilitate the biochemical processes
occurring within the bone. They occur at variable angles and depths
within the bone. Hence, when the machining occurs, the opening will
be varied and unpredictable resulting in a highly variable and
uncontrolled amount of BMP entering the surface of the bone.
In WO99/39,757 published Aug. 12, 1999, an osteoimplant is
disclosed which uses partially demineralized bone elements and
adjacent surface-exposed collagen to form chemical linkages to bond
the elements into a solid aggregate. It is noted in the Description
of the Preferred Embodiments, that "when prepared from bone derived
elements that are "only superficially dernineralized" that the
osteoimplant will possess a fairly high compression strength
approaching that of natural bone. FIG. 2 illustrates bone-derived
stacked sheets having a fully or partially demineralized outer
surface 21 with surface exposed collagen and a nondemineralized or
partially demineralized core 22. As noted in Example 1, the bone
sheets approximately 1.5 mm thick were placed in a 0.6N HCl
solution for 1.5 hours with constant stirring, washed in water for
5 minutes and soaked for 1.5 hours in phosphate buffered saline. In
Example 3 the bone-derived sheets from cortical bone were treated
for 10 minutes in 0.6N HCl to expose surface collagen. Bone cubes
derived from human cancerous bone were treated to expose surface
collagen at the outer borders of the cube. In Example 4, human
cortical bone-derived sheets approximately 1 mm thick were surface
demineralized for 15 minutes in 0.6N HCl and in Example 5, human
cortical bone derived sheets approximately 2 mm thick were surface
demineralized for 1 hour in 06N HCl.
U.S. Pat. No. 5,899,939, issued May, 1999, to the same inventor as
the foreign patent noted in the paragraph above, discloses a bone
derived implant made up of one or more layers of fully mineralized
or partially demineralized cortical bone, and optionally one or
more layers of some other material. The layers of the implant are
assembled into a unitary structure to provide an implant.
In U.S. Pat. No. 5,861,167, issued Jan. 19, 1999, a tooth root is
shown to have selective parts of the surface removed by acid to
improve subsequent attachment of the tooth in conjunction with
periodontal surgery. Similarly U.S. Pat. No. 5,455,041 utilized
treatment by demineralizing the tooth root surface with citric acid
applied for one minute to effect reattachment of collagen fibers to
the root surface and adding growth factors onto the surface of the
demineralized root
Partial demineralization of bone is also disclosed in the Journal
of Surgical Research Vol. 59, pages 614-620 (1995) in the article
Sterilization of Partially Demineralized Bone Matrix: The Effects
of Different Sterilization Techniques on Osteogenetic Properties
where particles of bone of 500 microns were treated for 24 hours at
4 degrees C with 0.6 N HC1 with the extent of decalcification
determined to be 20% and placed in the bone site. New bone
formation was noted after the passage of six weeks.
In French Patent Applications Numbers 2,582,517 and 2,582,518
treatment of fragments of bones taken from animals, primarily
cattle were partially demineralized and tanned with glutaraldehyde.
The bone elements to be implanted are cut to the desired shape from
an ox bone which has been subjected to a treatment comprising a
degreasing step with an organic solvent such as ethanol, a
demineralization step with a calcium extraction agent such as
hydrochloric acid and tanning with glutaraldehyde and subsequent
washings. Similar demineralization of bone is shown in U.S. Pat.
No. 5,585,116 issued Dec. 17, 1996. This patent also notes that it
is known that partial demineralization facilitates integration of a
bone graft. This is accordingly followed by different complementary
steps which are intended either to deproteinize the bone completely
or to act on the nature of the proteins which then remain linked
within the bone matrix or else to increase this proportion of
proteins.
It is desirable to make the surface of the bone more conductive to
receiving BMP's and other additives without losing the desirable
high mechanical strength properties of the cortical bone. It is
also desirable to leave most of the naturally occurring protein
intact in the bone in such a way as to expose just enough of the
bone surface to free the natural BMP's present on the surface.
Since demineralization also reduces the cross sectional area of the
bone construct, the bone construct must retain its shape and
structural integrity.
Accordingly, the prior art only partially addresses the problems
inherent in correcting surgical defects.
SUMMARY OF THE INVENTION
The present invention is directed toward the treatment of the
surface of cortical bone constructs to modify the surface by
removing a layer of the inorganic mineral hydroxyapatite material
leaving the mechanical properties of the bone constructs
substantially unchanged while providing a surface that allows the
addition of BMP's and other desirable additives to be introduced to
the surface and thereby enhance the healing rate of the cortical
bone in surgical procedures.
The subject formulation is a demineralized bone structure for
application to a bone defect site to promote new bone growth at the
site comprising a partially demineralized cortical bone structure,
said bone structure comprising a cross sectional surface are
ranging from 85% to 95% of the original bone surface area before
demineralization with the remaining partially demineralized
cortical bone structure comprising an outer demineralized layer
ranging in thickness from about 0.05% to about 0.14%. The structure
is designed to present the bone matrix and a demineralized surface
layer for reception of bone morphogenetic proteins (BMP) and other
desired additives. The macrostructure of the highly porous
demineralized surface layer serves both as an osteoconductive
matrix and to signal the patient's tissue and cells to initiate the
growth of new bone (osteoinduction).
It can be seen that the prior art has attempted to replicate to
some degree the present invention by flash demineralization of the
surface or fill dernineraliztion of the structure.
It is thus an object of the invention to provide a shaped bone
implant construct having a partially demineralized cortical bone
layer with an interior mineralized bone section to provide
compression strength to the implant bone construct.
It is an object of the invention to utilize a partially
demineralized shaped bone implant structure to approximate the
mechanical strength characteristics of natural bone to provide
overall strength and initial durability to the structure.
It is yet another object of the invention to provide a partially
demineralized shaped bone implant structure to provide a strong
implant structure of a predetermined shape and size for
implantation.
It is also an object of the invention to provide a bone derived
structure which can effective hold medical and biological
composition which promote new bone growth and accelerate
healing.
It is an additional object of the invention to use a BMP additive
in the demineralized layer of the bone structure.
It is an still additional object of the invention to use a soluble
silver additive in the demineralized layer of the bone
structure.
It is also an object off the invention to create a bone structure
which can be easily handled by the physician.
These and other objects, advantages, and novel features of the
present invention will become apparent when considered with the
teachings contained in the detailed disclosure which along with the
accompanying drawings constitute a part of this specification and
illustrate embodiments of the invention which together with the
description serve to explain the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of a partially demineralized rod or
dowel according to the invention;
FIG. 2 is a perspective view of a partially demineralized screw
according to the invention;
FIG. 3 is a perspective view of a partially demineralized anchor
according to the invention;
FIG. 4 is a perspective view of a partially demineralized wedge
according to the invention;
FIG. 5 is a perspective view of a partially demineralized fusion
ring according to the invention;
FIG. 6 is a perspective view of a partially demineralized composite
structure according to the invention;
FIG. 7 is a photograph of a 35.times.enlarged cross sectional view
of a partially demineralized rod treated with 0.6N HCl for 30
minutes;
FIG. 8 is a photograph of a 35.times. enlarged cross sectional view
of a partially demineralized rod treated with 0.6N HCl for 60
minutes;
FIG. 9 is a photograph of a 35.times. enlarged cross sectional view
of a partially demineralized rod treated with 0.6N HCl for 90
minutes;
FIG. 10 is a photograph of a 35.times. enlarged cross sectional
view of a partially demineralized rod treated with 0.6N HCl for 120
minutes;
FIG. 11 is a photograph of a 35.times. enlarged cross sectional
view of a partially demineralized rod treated with 0.6N HC1 for 180
minutes;
FIG. 12 is a graph showing bending displacement in relation to acid
soak time; and
FIG. 13 is a graph showing weight loss during partial
demineraliation in relation to acid soak time.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed towards a treated partially
demineralized cortical bone construct which can be placed in a bone
defect area to heal bone defects. The term cortical bone construct
means any shaped bone device such as rods, pins, dowels, screws,
plates, wedges, fusion rings, intervertaebral spacers and composite
assemblies. The aforementioned listing is exemplary only and is not
to construed as restrictive.
The preferred embodiment and the best mode as shown in FIGS. 1 and
7-11 and shows a cylindrical cortical bone construct 10 with its
surface 12 modified by acid treatment to remove a layer of the
inorganic, mineral, hydroxyapatite bone material in such a way as
to leave the mechanical properties substantially unchanged. While
the bone material is referred to as hydroxyapatite in this
application, in actuality the chemistry and structure of natural
bone mineral is different as natural bone mineral contains
carbonate ions, magnesium, sodium, hydrogen phosphate ions and
trace elements and a different crystalline structure than
hydroxyapatite.
The unique features of bone that makes it desirable as a surgical
material are, its ability to slowly resorb and be integrated into
the space it occupies while allowing the bodies own healing
mechanism to restore the repairing bone to its natural shape and
function by a mechanism known in the art as creeping substitution.
The second feature is the high mechanical strength arising from the
collagen fiber reinforced hydroxyapatite compound structure. The
creeping substitution mechanism, takes considerable time and some
forms of cortical bone in their natural, unmodified biological
state have been found to persist for over one year before
completely remodeling. Thus a means of accelerating the rate of
biointegration of cortical bone would improve the rate of healing
and benefit the recipient patient.
It is well known that bone contains osteoinductive elements known
as bone morphogenetic proteins (BMP). These BMP's are present
within the compound structure of cortical bone and are present at a
very low concentrations, e.g. 0.003%. Based upon the work of
Marshall Urist as shown in U.S. Pat. No. 4,294,753, issued Oct. 13,
1981 the proper demineraliation of cortical bone will expose the
BMP and present these osteoinductive factors to the surface of the
demineralized material rendering it significantly more
osteoinductive. The removal of the bone mineral leaves exposed
portions of collagen fibers allowing the addition of BMP's and
other desirable additives to be introduced to the demineralized
outer treated surface of the bone structure and thereby enhances
the healing rate of the cortical bone in surgical procedures. The
treatment process also exposes the naturally occurring BMP's at the
surface and renders the surface with biological properties similar
to full demineralized bone (DBM). The inner mass 14 of the bone
mineral of the shaped construct would be left intact to contain the
naturally occurring BMP's and trace elements as noted above. Such a
product would be beneficial in spinal fusion, fracture fixation and
simlar orthopaedic and neurological procedures where rapid healing
without loss of strength of implant is required. Partially
demineralized rods 16 as shown in FIG. 1 and FIGS. 7-11 will retain
various degrees of stiffness inversely proportional to the degree
of demineralization and retention of core mass. The partially
demineralized rods have a demineralized outer section 18 of exposed
collagen matrix and a cortical bone core 20.
Experiments conducted by the Applicants have discovered that the
surface of cortical bone constructs can be modified by acid
treatment to remove a layer of the inorganic, mineral,
hydroxyapatite material in such a way as to leave the mechanical
properties substantially unchanged or to provide a construct having
suitable compression and bending strength. This then allows the
addition of BMP's and other desirable additives to be introduced to
the surface and thereby enhance the healing rate of the cortical
bone in surgical procedures. The process also exposes the naturally
occurring BMP's near the surface and renders the surface with
biological properties similar to fully demineralized bone (DMB).
The inner mass of the bone construct would be left intact to
contain the naturally occurring BMP's.
It was found that when allograft cortical pins of 2.0 mm diameter
were treated as noted below in Example 1; and the pins were soaked
for 15 to 30 minutes in a 0.6N solution of HCl that there was
minimal loss of bending strength of the rod even when the diameter
of the rod was reduced from 3 to 5% and the outer layer was
demineratized. The demineralized layer ranged from about 0.05 to
about 0.08 mm reducing the mineralized portion diameter from 0.10
mm to 0.16 mm after 15 to 30 minutes of soaking in the 0.6N HCl
acid bath.
EXAMPLE 1
Allograft cortical bone pins were prepared by machining femoral or
tibial cortical bone. Pins were prepared with diameter of
approximately 2.0 mm and a length of 4 cm. The bulk bone segments
from which the pins were cut were chemically cleaned before
machining by soaking: 1) 30 minutes in an aqueous antibiotic
solution of Gentamycin. This reduces and eliminates any bioburden
introduced by handling the bone. 2) 30 minutes in an aqueous
detergent at 95.degree. F. using ultrasonic energy to enhance
penetration. This loosens and removes the lipid elements present in
and on the bone. 3) 60 minutes in a 70/30%v/v ethanol/water
solution. This further removes any lipid elements remaining after
the detergent wash in step 2, above. 4) The final cut pins were
given a final soak in a fresh solution of the ethanol/water
cleaning solution. 5) The pins were cut in half and then immersed
in a 0.6 N solution of Hydrochloric Acid (HCl). Half of each pin
was immersed for varying times and the other half was retained as
an untreated control. 6) The acid treatment was done at room
temperature, 23.degree. C. 7) Acid immersion was done for 30, 60,
90, 120 and 180 minutes. The pins were immersed in the acid
solution and agitated with gentle mechanical stirring. 8) After the
appropriate elapsed time the pins were removed, washed with
sterile, pure (USP Sterile) water until the wash discard was at
neutral pH. 9) The pins were then lyophilized and packaged in a
moisture permeable container.
For purpose of this example, the above treatments were done in a
laboratory setting. In a commercial process, the procedures would
be done in a sterile, clean room facility.
The acid treatment can be controlled to remove a small layer of the
bone mineral layer leaving a highly porous and compressible surface
layer while inducing no change to the inner mass of the construct.
By controlling the acid concentration, temperature and time of
exposure, a layer up to 0.06 mm can be removed and a layer 0.08 mm
demineralized and have the cortical pin experience substantially no
loss of mechanical properties as measured by a three-point bending
test. This is an unexpected result in that mass loss should have a
deleterious effect on bending resistance since the bending moment
of a cylindrical beam is a function of the third power of the
diameter.
Weight Loss, % Demineralization Time (n = 3) [0.6N HCl @ 23.degree.
C.] Average Std Dev 30 minutes 31.8 3.2 60 38.1 1.9 90 48.2 1.2 120
56.1 6.4 180 64.9 2.9
The thickness of the demineralized layer was also measured. For
each treated pin, the thickness of the demineralized layer was
measured six times by starting at the top of the bone traveling
clockwise approximately 60.degree.. The following data was
measured:
Thickness of Demineralized Layer Demineralization Time (mm) [0.6N
HCl @ 23.degree. C.] Average (n = 6) 30 minutes 0.08 60 0.11 90
0.14 120 0.17 180 0.25
The treated and control pins were subjected to a three-point
bending test. Force--displacement calculations were made from the
test results as are shown in FIG. 12. Bending displacement appears
to be directly proportional to the acid soak time after 30 minutes.
It is noteworthy that the bending displacement is equivalent for
the 30 minute soak time and the untreated control. Also note that
the 30 minute acid treatment did reduce the diameter of the pin
0.12 mm.
Scanning electron micrographs of the treated and control pins were
made and can be seen in the FIGS. 7, 8, 9, 10, and 11 reflecting
photographs of the same. It can be clearly seen that the Haversian
canals can be seen in the cross-section of the acid treated pins
and show the removal of the mineral layer at the surface at
35.times., revealing the open pores in the demineralized layer
exposed by the acid treatment.
This data demonstrates that surface demineralization can be
achieved to remove significant amounts of the surface mineral layer
without affecting the bulk mechanical strength.
Similar treatments were done for other machined cortical shapes
using 0.6N HCl at 23.degree. C. for 10 minutes: Example 2 Anterior
lumbar intervertebral fusion ring (FRA) Example 3 Posterior lumbar
intervertebral fusion block (PLIF) Example 4 Anterior cervical
fusion ring (ACF) Example 5 Allograft bone screw.
In all these examples, the surface of the machined cortical shape
was modified without loss of the key details and dimensions
machined into the surface.
The following shows the diameter change, the change in surface
morphology, and the size of the dernineralized layers in
cylindrical pins that were demineralized in 0.6N HCl in 30, 60, 90,
120, and 180 minutes.
1. Diameter Change
The diameter of each pin was measured in 3 places along the pin.
The measurements were recorded on the length of the photograph at
1.5 cm, 6.5 cm, and 11.5 cm on the pin. Each measurement is
recorded in the tables below. The bottom column in each "difference
between the treated and untreated pins" is the actual size
difference. The pin was magnified .times.35 so that the
measurements were each divided by 35 to arrive at the actual
difference diameter change.
Left Side Middle Right Side Pin 1-30 minute soak Untreated: Pin
1-B1 Measurement 6.6 cm 6.4 cm 6.5 cm Treated: Pin 1-B2 Measurement
6.0 cm 6.0 cm 6.2 cm Difference between the treated and untreated
pins Measurement 0.6 cm 0.4 cm 0.3 cm Actual 0.017 cm 0.011 cm
0.009 cm Difference Pin 2-60 minute soak Untreated: Pin 2-A2
Measurement 6.9 cm 7.1 cm 6.5 cm Treated: Pin 2-A2 Measurement 6.3
cm 6.3 cm 6.2 cm Difference between the treated and untreated pins
Measurement 0.6 cm 0.8 cm 0.3 cm Actual 0.017 cm 0.023 cm 0.009 cm
difference Pin 3-90 minute soak Untreated: Pin 3-C1 Measurement 7.1
cm 7.1 cm 6.9 cm Treated: Pin 3-C2 Measurement 5.9 cm 5.6 cm 5.4 cm
Difference between the treated and untreated pins Measurement 1.2
cm 1.5 cm 1.5 cm Actual 0.034 cm 0.043 cm 0.043 cm difference Pin
4-120 minute soak Untreated: Pin 4-A1 Measurement 6.9 cm 6.8 cm 6.6
cm Treated: Pin 4-A2 Measurement 5.1 cm 5.2 cm 4.9 cm Difference
between the treated and untreated pins Measurement 1.8 cm 1.6 1.7
Actual 0.051 cm 0.046 cm 0.049 cm Difference Pin 5-180 minute soak
Untreated: Pin 5-A2 Measurement 6.9 cm 6.9 cm 6.7 cm Treated: Pin
5-A2 Measurement 5.3 cm 4.6 cm 5.0 cm Difference between the
treated and untreated pins Measurement 1.6 cm 2.3 cm 1.3 cm Actual
0.046 cm 0.066 cm 0.037 cm difference Average diameter change for
pin 1: 0.012 cm (0.12 mm) Average diameter change for pin 2: 0.016
cm (0.16 mm) Average diameter change for pin 3: 0.040 cm (0.40 mm)
Average diameter change for pin 4: 0.049 cm (0.49 mm) Average
diameter change for pin 5: 0.050 cm (0.50 mm)
2. Surface Morphology
The surfaces of the treated pins were compared to the surfaces of
the untreated pins.
Pin Number Surface Morphology 1-B1 Particles are held very tightly
together. There are small gaps in the bone. It looks somewhat
rigid. 1-B2 Looks looser than 1-B1. Very rough looking. Can see
loose particles. There are many holes in the bone. Appears to have
more dimension/depth than 1-B1. 2-A1 Particles are held tightly
together. There are many small gaps in the bone. 2-A2 There are
many loose particles. The gaps are wider than 2-A1. 3-C1 Very dense
and rigid-looking. Particles are held tightly together. 3-C2 Not as
dense as 3-C1. There are many small surface holes and a couple of
loose particles. 4-A1 Particles held tightly together. Surface
appears very rigid. 4-A2 Surface smoother than 4-A1. There are many
surface holes (some deep enough to see the next layer some just
forming). A couple of loose particles. 5-A1 Very dense and rigid.
Small gaps. 5-A2 Smoother than 5-A1. Many surface holes. Towards
the top of the slide, the bone appears bumpy. Gaps are wider than
in 5-A1.
3. Thickness of the Deminineralized Layer
For each treated pin, the thickness of the demineralized layer was
measured 6 times and the average per pin was calculated and
recorded. Note: The measurements started at the top of the bone and
recorded clockwise at approximately 60.degree. intervals. (A
magnifying glass with a cm ruler on it was used to measure the
demnineralized layer of each pin).
Pin Measurement Number Average Number 1 2 3 4 5 6 Thickness 1-B2
0.09 mm 0.09 mm 0.06 mm 0.11 mm 0.06 mm 0.09 mm 0.08 mm 2-A2 0.11
mm 0.09 mm 0.09 mm 0.11 mm 0.14 mm 0.11 mm 0.11 mm 3-C2 0.14 mm
0.06 mm 0.03 mm 0.17 mm 0.29 mm 0.14 mm 0.14 mm 4-A2 0.17 mm 0.20
mm 0.20 mm 0.17 mm 0.11 mm 0.14 mm 0.17 mm 5-A2 0.26 mm 0.23 mm
0.20 mm 0.23 mm 0.29 mm 0.29 mm 0.25 mm
4. Results
The length of acid soak has an effect on the diameter of the pin.
While longer the pin is soaked in 0.6N HCl, the more the diameter
changes in size (the diameter gets smaller), a relatively constant
diameter was reached after the 120 minutes of soak in the HCC. The
average diameter change for the pin soaked for 30 minutes was 0.12
mm; for 60 minutes was 0.16 mm; for 90 minutes was 0.40 mm; and for
120 minutes was 0.49 mm and 180 minutes was 0.50 mm. The
cross-section slides show that while the diameter of the pins
decreased at an increased amount from soak minutes 60 to 90
lessening from soak minutes 90 to 120, it remaining substantially
constant thereafter. The thickness of the demineralized layer
increased almost linearly.
The surface morphology was also affected by the acid soaks. All the
pins were viewed under a magnification of 100.times.. The slides of
the untreated pins looked rigid, the particles were tightly held
into place making the bone to appear dense, and there were small
gaps on some sections of the bones. The slides of the treated pins
looked completely different than the untreated pins. The
treated-pin slides show loose particles, surface holes, widened
gaps, and the bones appear to be less dense.
Overall, the length of acid soak time affects the three areas
tested in this study: 1. The longer the pin soaks in 0.6N HCl, the
actual diameter of the pin decreases up until 120 minutes of acid
soak. 2. The longer the pin is in the acid soak, the thickness of
the demineralized layer on the bone increases and the core
mineralized portion decreases. 3. The acid also has an effect on
the surface morphology of the bone. It changes the surface
morphology from appearing very dense and rigid (when untreated) to
having loose particles and becoming somewhat smoother (when
treated).
It is valuable to add soluble silver (e.g. AgNO.sub.3) to the
surface treated cortical bone structure. This will provide
biostatic properties to the construct, i.e., it will inhibit any
growth of microorganisms which may be resident on the surface of
the cortical tissue or adjacent to it in the surrounding tissue. At
sufficiently high concentrations, the silver cation will be fully
biocidal. Thus, silver ranging from 10 to 10,000 parts per million
may be used.
It is also envisioned to add soluble silver to the surface after
treatment to provide biostatic properties inhibiting any growth of
microorganisms which may be resident on the surface of the cortical
tissue or adjacent to it in the surrounding tissue. Silver which
can be added is can be taken from a group consisting of silver
nitrate and other soluble or slightly soluble silver compounds such
as silver chloride, silver oxide, silver sulphate, silver
phosphate, silver acetate, silver perchlorate or silver
tartrate.
It is also possible to add one or more rhBMP's to the surface of
the treated bone shape by soaking and being able to use a
significantly lower concentration of the rare and expensive
recombinant human BMP to achieve the same acceleration of
biointegration. The addition of other useful treatment agents such
as vitamins, hormones, antibiotics, antiviral and other therapeutic
agents could also be added to the surface modified layer. BMP
directs the differentiation of pluripotential mesenchymal cells
into osteoprogenitor cells which form osteoblasts. The ability of
freeze dried demineralized cortical bone to facilitate this bone
induction principle using BMP present in the bone is well known in
the art. However, the amount of BMP varies in the bone depending on
the age of the bone donor and the bone processing. Sterilization is
an additional problem in processing human bone for medical use as
boiling, autoclaving or irradiation over 2.0 Mrads is sufficient to
destroy or alter the BMP present in the bone matrix.
The time, temperature and acid concentration can be adjusted to
achieve a set of process conditions that will give the same
physical result as the above noted examples. Temperature could be
lowered to 4.degree. C. and allow the process time to increase to
one hour (a four fold increase in process time). Temperatures much
above 30.degree. C. will result in too rapid a rate of
hydroxyapatite removal and result in a highly variable shape.
Conditions could be adjusted to use acid concentrations from about
0.1N to about 2.0N HCl. Lower concentrations will result in a very
slow rate of mineral layer removal, not conducive to a commercial
process. Higher concentrations will result in a too rapid rate of
mineral removal and to a highly varied and uncontrolled surface.
Other acids could be used; sulfuric, phosphoric or other mineral
acids, organic acids such as acetic; cheating agents such as
ethylene diamine tetra acetic acid or other weak acids would also
be suitable.
Any number of medically useful substances can be incorporated in
the invention by adding the substances to the composition at any
steps in the mixing process or directly to the final composition.
Such substances include collagen and insoluble collagen
derivatives, hydroxyapatite and soluble sods and/or liquids
dissolved therein. Also included are antiviricides such as those
effective against HIV and hepatitis; antimicrobial and/or
antibiotics such as erythromycin, bacitracin, neomycin, penicillin,
polymyxin B, tetracycline, viomycin, chloromycetin and
streptomycin, cefazolin, ampicillin, azactam, tobramycin,
clindamycin and gentamycin. It is also envisioned that amino acids,
peptides, vitamins, co-factors for protein synthesis; hormones;
endocrine tissue or tissue fragments; synthesizers; enzymes such as
collagenase, peptidases, oxidases; polymer cell scaffolds with
parenchymal cells; angiogenic drugs and polymeric carriers
containing such drugs; collagen lattices; biocompatible surface
active agents, antigenic agents; cytoskeletal agents; cartilage
fragments, living cells such as chondrocytes, bone marrow cells,
mesenchymal stem cells, natural extracts, tissue transplants,
bioadhesives, transforming growth factor (TGF-beta), insulin-like
growthfactor (IGF-1); growth hormones such as somatotropin; bone
digesters; antitumor agents; fibronectin; cellular attractants and
attachment agents; immuno-suppressants; permeation enhancers, e.g.
fatty acid esters such as laureate, myristate and stearate
monoesters of polyethylene glycol, enamine derivatives, alpha-keto
aldehydes can be added to the composition
All products can also be done in an aseptic environment to maintain
a sterile final product or sterilized after production. The
cortical bone structure is then placed in a moisture permeable
inner container which is placed in a moisture barrier outer
container.
The principles, preferred embodiments and modes of operation of the
present invention have been described in the foregoing
specification. However, the invention should not be construed as
limited to the particular embodiments which have been described
above. Instead, the embodiments described here should be regarded
as illustrative rather than restrictive. Variations and changes may
be made by others without departing from the scope of the present
invention as defined by the following claims:
* * * * *